A major challenge in accomplishing genomic precision medicine is the observation that genotype does not always predict phenotype. Incomplete penetrance and variable expressivity represent two examples of this phenotypic variability. This complexity is well-illustrated by human mutations in PROK2 (encoding prokineticin 2) and PROKR2 (prokineticin receptor 2), that cause human Isolated GnRH (gonadotropin-releasing hormone) Deficiency (IGD). Prokineticin 2 signaling is a master regulator of mammalian reproduction and is associated with neurodevelopmental and neuroendocrine forms of IGD in humans (Kallmann syndrome, and normosmic IGD, respectively). In addition to this physiologic complexity, PROK2/R2 mutations are predominantly missense mutations that exhibit either reduced penetrance and/or variable expressivity. The genetic basis of this variability is not fully understood but allelic heterogeneity and modifier genes have been postulated to account for this variability. In Specific Aim 1, allelic heterogeneity of human PROKR2 mutations will be examined in vitro, using PROK2 signaling assays and allele specific expression studies. Whole exome sequencing and SNP genotyping will identify modifier genes that contribute to disease penetrance and expressivity. PROK2 is also implicated in diverse physiologic processes such as glucose metabolism, energy balance, food intake, circadian rhythms, pain modulation, neutrophil chemotaxis and angiogenesis. The human relevance of these pleotropic functions of PROK2 is yet to be established. In Specific Aim 2, phenomic dissection of humans with PROK2/R2 mutations will be undertaken to define the pleotropic phenotypes relating to PROK2/R2 mutations in humans. IGD patients as well as control individuals in a tertiary hospital biobank population will be studied. Reproductive phenotypes and non-reproductive phenotypes will be charted. Targeted phenotyping will include assessment of glucose homeostasis and pain modulation in both groups. A phenome-wide association study will be conducted in the biobank subjects harboring PROK2/R2 mutations. In Specific Aim 3, using a novel PROKR2-cre mouse model, transcriptomic studies of PROKR2 and GnRH neurons will be done at various developmental timepoints using temporal inhibition of PROKR2 using chemogenetics. Sexual maturation and fertility of these mice after temporal PROKR2 inhibition will also be examined. The top genes that are differentially expressed in the murine transcriptomic experiments will be translationally validated by assessing whether these genes emerge as modifier genes in the human whole exome sequencing studies. The studies in this proposal have the potential to unravel novel genetic mechanisms of disease penetrance relating to the PROK2 pathway mutations, uncover potential pleotropic human phenotypes associated with mutations in this pathway and help deliver personalized medicine to humans with reproductive disorders and infertility.